Transcription factor HNF4α2 promotes osteogenesis and prevents bone abnormalities in mice with renal osteodystrophy.

Renal osteodystrophy (ROD) is a disorder of bone metabolism that affects virtually all patients with chronic kidney disease (CKD) and is associated with adverse clinical outcomes including fractures, cardiovascular events, and death. In this study, we showed that hepatocyte nuclear factor 4α (HNF4α), a transcription factor mostly expressed in the liver, is also expressed in bone, and that osseous HNF4α expression was dramatically reduced in patients and mice with ROD. Osteoblast-specific deletion of Hnf4α resulted in impaired osteogenesis in cells and mice. Using multi-omics analyses of bones and cells lacking or overexpressing Hnf4α1 and Hnf4α2, we showed that HNF4α2 is the main osseous Hnf4α isoform that regulates osteogenesis, cell metabolism, and cell death. As a result, osteoblast-specific overexpression of Hnf4α2 prevented bone loss in mice with CKD. Our results showed that HNF4α2 is a transcriptional regulator of osteogenesis, implicated in the development of ROD.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

Transcriptomics: a Solution for Renal Osteodystrophy?

PURPOSE OF REVIEW: The molecular mechanisms of the bone disease associated with chronic kidney disease (CKD), called renal osteodystrophy (ROD), are poorly understood. New transcriptomics technologies may provide clinically relevant insights into the pathogenesis of ROD. This review summarizes current progress and limitations in the study and treatment of ROD, and in transcriptomics analyses of skeletal tissues. RECENT FINDINGS: ROD is characterized by poor bone quality and strength leading to increased risk of fracture. Recent studies indicate permanent alterations in bone cell populations during ROD. Single-cell transcriptomics analyses, successful at identifying specialized cell subpopulations in bone, have not yet been performed in ROD. ROD is a widespread poorly understood bone disease with limited treatment options. Transcriptomics analyses of bone are needed to identify the bone cell subtypes and their role in the pathogenesis of ROD, and to develop adequate diagnosis and treatment strategies.

The predictive value of Klotho polymorphism, in addition to classical markers of CKD-MBD, for left ventricular hypertrophy in haemodialysis patients.

PURPOSE: Cardiovascular events are the major reasons for mortality in haemodialysis patients. Fibroblast growth factor 23 (FGF23), Klotho protein and G-395A Klotho gene polymorphism have been associated with effects on the cardiovascular system. Our study investigates the interrelationship between Klotho protein gene variations, mineral-bone metabolism and left ventricular hypertrophy in patients undergoing chronic haemodialysis programme. MATERIALS AND METHODS: Patients (n = 142) were genotyped for G-395A Klotho gene. Components of mineral-bone metabolism, classical and non-classical (FGF23, Klotho and vitamin D) as well as echocardiographic examination were determined. Predictive models were designed to determine the significance of Klotho gene variations and mineral-bone metabolism components for left ventricle hypertrophy (LVH). RESULTS: A-allele carriers were longer on haemodialysis (p = 0.033), and had higher phosphorus levels (p = 0.016) while the level of Klotho protein was significantly lower (p = 0.001) compared to non-A-allele carriers. The best gains were achieved upon addition of allele A, and all three new markers; the AUC made significant improvement from 0.596 to 0.806 (p < 0.001), and improved net reclassification for 82.1% (95% CI 42.9-121.3%). CONCLUSIONS: The genetic background of A-allele carriers of the G-395A Klotho gene polymorphism increases the susceptibility patients to haemodialysis. A-allele carriers are at a higher risk for the development of cardiovascular complications. The addition of non-classical to classical mineral metabolism components improves prediction power to LVH.

Effect of ovariectomy on the progression of chronic kidney disease-mineral bone disorder (CKD-MBD) in female Cy/+ rats.

Male Cy/+ rats have shown a relatively consistent pattern of progressive kidney disease development that displays multiple key features of late stage chronic kidney disease-mineral bone disorder (CKD-MBD), specifically the development of cortical bone porosity. However, progression of disease in female Cy/+ rats, assessed in limited studies, is more heterogeneous and to date has failed to show development of the CKD-MBD phenotype, thus limiting their use as a practical model of progressive CKD-MBD. Animal and human studies suggest that estrogen may be protective against kidney disease in addition to its established protective effect on bone. Therefore, in this study, we aimed to determine the effect of ovariectomy (OVX) on the biochemical and skeletal manifestations of CKD-MBD in Cy/+ female rats. We hypothesized that OVX would accelerate development of the biochemical and skeletal features of CKD-MBD in female Cy/+ rats, similar to those seen in male Cy/+ rats. Female Cy/+ rats underwent OVX (n = 8) or Sham (n = 8) surgery at 15 weeks of age. Blood was collected every 5 weeks post-surgery until 35 weeks of age, when the rats underwent a 4-day metabolic balance, and the tibia and final blood were collected at the time of sacrifice. OVX produced the expected changes in trabecular and cortical parameters consistent with post-menopausal disease, and negative phosphorus balance compared with Sham. However, indicators of CKD-MBD were similar between OVX and Sham (similar kidney weight, plasma blood urea nitrogen, creatinine, creatinine clearance, phosphorus, calcium, parathyroid hormone, and no cortical porosity). Contrary to our hypothesis, OVX did not produce evidence of development of the CKD-MBD phenotype in female Cy/+ rats.

Fibroblast Growth Factor 23 Genotype and Cardiovascular Disease in Patients Undergoing Hemodialysis.

BACKGROUND: Elevated serum concentrations of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality in patients with chronic kidney disease and those undergoing dialysis. OBJECTIVES: We tested the hypotheses that polymorphisms in FGF23, its co-receptor alpha-klotho (KL), and/or FGF23 receptors (FGFR) are associated with cardiovascular events and/or mortality. METHODS: We used 1,494 DNA samples collected at baseline from the Evaluation of Cinacalcet HCl Therapy to Lower Cardiovascular Events Trial, in which patients were randomized to the calcimimetic cinacalcet or placebo for the treatment of secondary hyperparathyroidism. We analyzed European and African Ancestry samples separately and then combined summary statistics to perform a meta-analysis. We evaluated single-nucleotide polymorphisms (SNPs) in FGF23, KL, and FGFR4 as the key exposures of interest in proportional hazards (Cox) regression models using adjudicated endpoints (all-cause and cardiovascular mortality, sudden cardiac death, and heart failure [HF]) as the outcomes of interest. RESULTS: rs11063112 in FGF23 was associated with cardiovascular mortality (risk allele = A, hazard ratio [HR] 1.32, meta-p value = 0.004) and HF (HR 1.40, meta-p value = 0.007). No statistically significant associations were observed between FGF23 rs13312789 and SNPs in FGFR4 or KL genes and the outcomes of interest. CONCLUSIONS: rs11063112 was associated with HF and cardiovascular mortality in patients receiving dialysis with moderate to severe secondary hyperparathyroidism.

Update on Chronic Kidney Disease Mineral and Bone Disorder in Cardiovascular Disease.

Chronic kidney disease mineral and bone disorder (MBD) encompasses changes in mineral ion and vitamin D metabolism that are widespread in the setting of chronic kidney disease and end-stage renal disease. MBD components associate with cardiovascular disease in many epidemiologic studies. Through impacts on hypertension, activation of the renin-angiotensin-aldosterone system, vascular calcification, endothelial function, and cardiac remodeling and conduction, MBD may be a direct and targetable cause of cardiovascular disease. However, assessment and treatment of MBD is rife with challenges owing to biological tensions between its many components, such as calcium and phosphorus with their regulatory hormones fibroblast growth factor 23 and parathyroid hormone; fibroblast growth factor 23 with its co-receptor klotho; and vitamin D with control of calcium and phosphorus. These complex interactions between MBD components hinder the simple translation to clinical trials, which ultimately are needed to prove the benefits of treating MBD. Deeper investigation using precision medicine tools and principles, including genomics and individualized risk assessment and therapy, may help move the field closer toward clinical applications. This review provides a high-level overview of conventional and precision epidemiology in MBD, potential mechanisms of cardiovascular disease pathogenesis, and guiding therapeutic principles for established and emerging treatments.

Systemic Activation of Activin A Signaling Causes Chronic Kidney Disease-Mineral Bone Disorder.

The high cardiovascular mortality associated with chronic kidney disease (CKD) is caused in part by the CKD-mineral bone disorder (CKD-MBD) syndrome. The CKD-MBD consists of skeletal, vascular and cardiac pathology caused by metabolic derangements produced by kidney disease. The prevalence of osteopenia/osteoporosis resulting from the skeletal component of the CKD-MBD, renal osteodystrophy (ROD), in patients with CKD exceeds that of the general population and is a major public health concern. That CKD is associated with compromised bone health is widely accepted, yet the mechanisms underlying impaired bone metabolism in CKD are not fully understood. Therefore, clarification of the molecular mechanisms by which CKD produces ROD is of crucial significance. We have shown that activin A, a member of the transforming growth factor (TGF)-ß super family, is an important positive regulator of receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis with Smad-mediated signaling being crucial for inducing osteoclast development and function. Recently, we have demonstrated systemic activation of activin receptors and activin A levels in CKD mouse models, such as diabetic CKD and Alport (AL) syndrome. In these CKD mouse models, bone remodeling caused by increased osteoclast numbers and activated osteoclastic bone resorption was observed and treatment with an activin receptor ligand trap repaired CKD-induced-osteoclastic bone resorption and stimulated individual osteoblastic bone formation, irrespective of parathyroid hormone (PTH) elevation. These findings have opened a new field for exploring mechanisms of activin A-enhanced osteoclast formation and function in CKD. Activin A appears to be a strong candidate for CKD-induced high-turnover ROD. Therefore, the treatment with the decoy receptor for activin A might be a good candidate for treatment for CKD-induced osteopenia or osteoporosis, indicating that the new findings from in these studies will lead to the identification of novel therapeutic targets for CKD-related and osteopenia and osteoporosis in general. In this review, we describe the impact of CKD-induced Smad signaling in osteoclasts, osteoblasts and vascular cells in CKD.

Associations between single nucleotide polymorphisms in the calcium sensing receptor and chronic kidney disease-mineral and bone disorder in cats.

Feline chronic kidney disease (CKD) is associated with high variability in severity of CKD-mineral and bone disorder (CKD-MBD). The calcium sensing receptor (CaSR) regulates circulating parathyroid hormone (PTH) and calcium concentrations. Single nucleotide polymorphisms (SNPs) in the CaSR are associated with severity of secondary renal hyperparathyroidism and total calcium concentrations in human patients receiving haemodialysis. The objective of this study was to explore associations between polymorphisms in the feline CaSR (fCaSR) and biochemical changes observed in CKD-MBD. Client owned cats (≥9years) were retrospectively included. SNP discovery was performed in 20 cats with azotaemic CKD and normal or dysregulated calcium concentrations. Non-pedigree cats (n=192) (125 with azotaemic CKD and 66 healthy), Persians (n=40) and Burmese (n=25) were genotyped for all identified SNPs using KASP. Biochemical parameters from the date of CKD diagnosis or from first visit to the clinic (healthy cats) were used. Associations between genotype and ionized calcium, total calcium, phosphate, PTH and FGF-23 were performed for non-pedigree cats using logistic regression. Sequence alignment against the fCaSR sequence revealed eight novel exonic SNPs. KASP genotyping had high accuracy (99.6%) and a low failure rate (<6%) for all SNPs. Allele frequencies varied between breeds. In non-pedigree cats, one synonymous SNP CaSR:c.1269G>A was associated with logPTH concentration (adjusted for plasma creatinine concentration), with a recessive model having the best fit (G/G vs A/A-G/A, P=0.031). Genetic variation in the fCaSR is unlikely to explain the majority of the variability in presence and severity of CKD-MBD in cats.

The activin receptor is stimulated in the skeleton, vasculature, heart, and kidney during chronic kidney disease.

We examined activin receptor type IIA (ActRIIA) activation in chronic kidney disease (CKD) by signal analysis and inhibition in mice with Alport syndrome using the ActRIIA ligand trap RAP-011 initiated in 75-day-old Alport mice. At 200 days of age, there was severe CKD and associated Mineral and Bone Disorder (CKD-MBD), consisting of osteodystrophy, vascular calcification, cardiac hypertrophy, hyperphosphatemia, hyperparathyroidism, elevated FGF23, and reduced klotho. The CKD-induced bone resorption and osteoblast dysfunction was reversed, and bone formation was increased by RAP-011. ActRIIA inhibition prevented the formation of calcium apatite deposits in the aortic adventitia and tunica media and significantly decreased the mean aortic calcium concentration from 0.59 in untreated to 0.36 mg/g in treated Alport mice. Aortic ActRIIA stimulation in untreated mice increased p-Smad2 levels and the transcription of sm22α and αSMA. ActRIIA inhibition reversed aortic expression of the osteoblast transition markers Runx2 and osterix. Heart weight was significantly increased by 26% in untreated mice but remained normal during RAP-011 treatment. In 150-day-old mice, GFR was significantly reduced by 55%, but only by 30% in the RAP-011-treated group. In 200-day-old mice, the mean BUN was 100 mg/dl in untreated mice compared to 60 mg/dl in the treated group. In the kidneys of 200-day-old mice, ActRIIA and p-Smad2 were induced and MCP-1, fibronectin, and interstitial fibrosis were stimulated; all were attenuated by RAP-011 treatment. Hence, the activation of ActRIIA signaling during early CKD contributes to the CKD-MBD components of osteodystrophy and cardiovascular disease and to renal fibrosis. Thus, the inhibition of ActRIIA signaling is efficacious in improving and delaying CKD-MBD in this model of Alport syndrome.

ADAM17, a New Player in the Pathogenesis of Chronic Kidney Disease-Mineral and Bone Disorder.

The triad composed by α-Klotho, fibroblast growth factor-23, and its receptor are involved in the pathogenesis of chronic kidney disease-mineral and bone disorder. A disintegrin and metalloproteinase 17 (ADAM17) is a metalloproteinase causing the proteolytic shedding of α-Klotho from the cell membrane, and its role in chronic kidney disease-mineral and bone disorder is not yet known. We studied the circulating levels of the above-mentioned mediators in patients with secondary hyperparathyroidism due to uremia, compared to control subjects, as well as in patients with primary hyperparathyroidism. We also measured the immunofluorescence pattern of the relevant tissue proteins in specimens obtained from patients undergoing parathyroid surgery for secondary compared to primary hyperparathyroidism. Results showed that α-Klotho tissue levels are reduced, in the presence of increased ADAM17 tissue levels. In addition, we showed increased serum levels of the main product of ADAM17 proteolytic activity, tumor necrosis factor-α. Thus, we found a paradoxical situation, in secondary compared to primary hyperparathyroidism, that is, that in the face of increased tumor necrosis factor-α in circulation, both soluble and tissue α-Klotho are reduced significantly, despite increased tissue ADAM17. In conclusion, tissue and serum levels of α-Klotho seem to have become independent from the regulation induced by ADAM17, which constitutes therefore another tassel in the impaired α-Klotho-FGF23 receptor axis present in uremia.

Calcium-Sensing Receptor Genotype and Response to Cinacalcet in Patients Undergoing Hemodialysis.

BACKGROUND AND OBJECTIVES: We tested the hypothesis that single nucleotide polymorphisms (SNPs) in the calcium-sensing receptor (CASR) alter the response to the calcimimetic cinacalcet. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed DNA samples in the Evaluation of Cinacalcet HCl Therapy to Lower Cardiovascular Events (EVOLVE) trial, a randomized trial comparing cinacalcet to placebo on a background of usual care. Of the 3883 patients randomized, 1919 (49%) consented to DNA collection, and samples from 1852 participants were genotyped for 18 CASR polymorphisms. The European ancestry (EA; n=1067) and African ancestry (AfAn; n=405) groups were assessed separately. SNPs in CASR were tested for their association with biochemical measures of mineral metabolism at baseline, percent change from baseline to 20 weeks, and risk of clinical fracture as dependent variables. RESULTS: There were modest associations of CASR SNPs with increased baseline serum parathyroid hormone and bone alkaline phosphatase primarily with the minor allele in the EA group (all P≤0.03), but not in the AfAn sample. In contrast, there was a modest association of decreased baseline serum calcium and FGF23 with CASR SNPs (P=0.04) primarily with the minor allele in the AfAn but not in the EA sample. The minor allele of two SNPs was associated with decreased percent reduction in parathyroid hormone from baseline to 20 weeks in the EA population (P<0.04) and this was not altered with cinacalcet. In both EA and AfAn, the same SNP (rs9740) was associated with decreased calcium with cinacalcet treatment (EA and AfAn P≤0.03). Three SNPs in high linkage disequilibrium were associated with a higher risk of clinical fracture that was attenuated by cinacalcet treatment in the EA sample (P<0.04). CONCLUSIONS: These modest associations, if validated, may provide explanations for differences in CKD-mineral bone disorder observed in EA and AfAn populations, and for differential biochemical responses to calcimimetics.

[ARTICLE WITHDRAWN] Satb1 promotes osteoclastogenesis by recruiting CBP to upregulate miR-223 expression in chronic kidney disease-mineral and bone disorder.

THIS ARTICLE WAS WITHDRAWN BY THE PUBLISHER IN OCT 2021

microRNAs in the pathophysiology of CKD-MBD: Biomarkers and innovative drugs.

microRNAs comprise a novel class of endogenous small non-coding RNAs that have been shown to be implicated in both vascular damage and bone pathophysiology. Chronic kidney disease-mineral bone disorder (CKD-MBD) is characterized by vessel and bone damage secondary to progressive loss of kidney function. In this review, we will describe how several microRNAs have been implicated, in recent years, in cellular and animal models of CKD-MBD, and have been very recently shown to be deregulated in patients with CKD. Particular emphasis has been placed on the endothelial-specific miR-126, a potential biomarker of endothelial dysfunction, and miR-155 and miR-223, which play a role in both vascular smooth muscle cells and osteoclasts, with an impact on the vascular calcification and osteoporosis process. Finally, as these microRNAs may constitute useful targets to prevent or treat complications of CKD-MBD, we will discuss their potential as innovative drugs, describe how they could be delivered in a timely and specific way to vessels and bone by using the most recent techniques such as nanotechnology, viral vectors or CRISPR gene targeting.

Only minor differences in renal osteodystrophy features between wild-type and sclerostin knockout mice with chronic kidney disease.

Renal osteodystrophy affects the majority of patients with advanced chronic kidney disease (CKD) and is characterized by progressive bone loss. This study evaluated the effects of sclerostin knockout on bone in a murine model of severe, surgically induced CKD in both sclerostin knockout and wild-type mice. Mice of both genotypes with normal kidney function served as controls. Tibiae were analyzed using micro-computed tomography, and lumbar vertebrae were analyzed by histomorphometry. Results were tested for statistical significance by 2-way ANOVA to investigate whether bone of the knockout mice reacted differently to CKD compared with bone of wild-type mice. In the tibiae, there was no difference after creation of CKD between wild-type and knockout animals for cortical thickness or cross-sectional moment of inertia. Increases in cortical porosity induced by CKD differed significantly between genotypes in the tibial metaphysis but not in the diaphysis. In the trabecular compartment, no difference in reaction to CKD between genotypes was found for bone volume, trabecular number, trabecular thickness, and trabecular separation. In the lumbar vertebrae, significant differences in response to CKD between wild-type and knockout mice were seen for both bone volume and trabecular thickness. Osteoblast parameters did not differ significantly, whereas osteoclast numbers significantly increased in the wild-type but significantly decreased in knockout mice with CKD. No differences in response to CKD between genotypes were found for bone formation rate or mineral apposition rate. Thus, complete absence of sclerostin has only minor effects on CKD-induced bone loss in mice.

Association of vitamin D receptor gene polymorphisms with end-stage renal disease and the development of high-turnover renal osteodystrophy in a Chinese population.

Two single nucleotide polymorphisms (SNPs; TaqI and ApaI) in the vitamin D receptor (VDR) gene have been identified as risk factors for the progression of end-stage renal disease (ESRD). The purpose of our study was to confirm the reported association of these two SNPs with ESRD risk and progression of renal osteodystrophy in a Chinese Han population. A total of 452 ESRD patients and 904 matched-pair controls (based on age, gender, and body mass index) were included. Identification of VDR gene polymorphisms was performed using the polymerase chain reaction-restriction fragment length polymorphism method with TaqI and ApaI restriction enzymes. There was no association of the TaqI polymorphism with ESRD risk. However, significant associations were seen between ApaI (rs7975232) polymorphism and ESRD risk in the heterozygote model (AC/ AA; P = 0.002; OR = 1.4, 95%CI = 1.14-1.83), homozygote model (CC/AA; P = 0.007; OR = 1.8, 95%CI = 1.17-2.85) genotypes for rs7975232, allelic model (P < 0.001; OR = 1.4, 95%CI = 1.15-1.64), dominant model (P = 0.001; OR = 1.5, 95%CI = 1.19-1.87), and recessive model (P = 0.046; OR = 0.6, 95%CI = 0.42-1.00) between cases and healthy controls Moreover, we found a significant correlation between the genotype and allele distribution of ApaI and intact parathyroid hormone (iPTH) levels, where allele C carriers have increased iPTH levels. The ApaI polymorphism in the VDR gene appears to be a susceptibility locus for ESRD in Chinese individuals, and allele C carriers may have an increased risk of high-turnover renal osteodystrophy.

Therapeutic effect of CNP on renal osteodystrophy by antagonizing the FGF-23/MAPK pathway.

Renal osteodystrophy (ROD) is highly prevalent in chronic kidney disease (CKD). Because most patients with ROD are asymptomatic in the early stage and bone biopsy remains not a routine procedure in many clinical settings; therefore, several biochemical parameters may help to identify the existence of ROD. C-type natriuretic peptide (CNP) is considered as a positive regulator of bone formation. Both urinary excretion and renal expression of CNP are markedly up-regulated in the early stages of CKD, whereas they are still progressively declined accompanied by CKD progression, which invites speculation that the progressive decline of CNP may contribute, in part, to the pathogenesis of ROD. In addition, fibroblast growth factor (FGF)-23 is a bone-derived endocrine regulator of phosphate homeostasis. The elevation of serum FGF-23 has been recognized as a common feature in CKD to maintain normophosphatemia at the expense of declining 1,25-dihydroxyvitamin D values. Since the effects of CNP and FGF-23 on bone formation appear to oppose each other, it is reasonable to propose a direct interaction of their signaling pathways during the progression of ROD. CNP and FGF-23 act through a close or reciprocal pathway and are in agreement with recent studies demonstrating a down-regulatory role of the mitogen-activated protein kinase activity by CNP. The specific node may act at the level of RAF-1 through the activation of cyclic guanosine monophosphate-dependent protein kinases II.

Primary osteoblast-like cells from patients with end-stage kidney disease reflect gene expression, proliferation, and mineralization characteristics ex vivo.

Osteocytes regulate bone turnover and mineralization in chronic kidney disease. As osteocytes are derived from osteoblasts, alterations in osteoblast function may regulate osteoblast maturation, osteocytic transition, bone turnover, and skeletal mineralization. Thus, primary osteoblast-like cells were cultured from bone chips obtained from 24 pediatric ESKD patients. RNA expression in cultured cells was compared with RNA expression in cells from healthy individuals, to RNA expression in the bone core itself, and to parameters of bone histomorphometry. Proliferation and mineralization rates of patient cells were compared with rates in healthy control cells. Associations were observed between bone osteoid accumulation, as assessed by bone histomorphometry, and bone core RNA expression of osterix, matrix gla protein, parathyroid hormone receptor 1, and RANKL. Gene expression of osteoblast markers was increased in cells from ESKD patients and signaling genes including Cyp24A1, Cyp27B1, VDR, and NHERF1 correlated between cells and bone cores. Cells from patients with high turnover renal osteodystrophy proliferated more rapidly and mineralized more slowly than did cells from healthy controls. Thus, primary osteoblasts obtained from patients with ESKD retain changes in gene expression ex vivo that are also observed in bone core specimens. Evaluation of these cells in vitro may provide further insights into the abnormal bone biology that persists, despite current therapies, in patients with ESKD.

Role of TGF-ß in a mouse model of high turnover renal osteodystrophy.

Altered bone turnover is a key pathologic feature of chronic kidney disease-mineral and bone disorder (CKD-MBD). Expression of TGF-ß1, a known regulator of bone turnover, is increased in bone biopsies from individuals with CKD. Similarly, TGF-ß1 mRNA and downstream signaling is increased in bones from jck mice, a model of high-turnover renal osteodystrophy. A neutralizing anti-TGF-ß antibody (1D11) was used to explore TGF-ß's role in renal osteodystrophy. 1D11 administration to jck significantly attenuated elevated serum osteocalcin and type I collagen C-telopeptides. Histomorphometric analysis indicated that 1D11 administration increased bone volume and suppressed the elevated bone turnover in a dose-dependent manner. These effects were associated with reductions in osteoblast and osteoclast surface areas. Micro-computed tomography (µCT) confirmed the observed increase in trabecular bone volume and demonstrated improvements in trabecular architecture and increased cortical thickness. 1D11 administration was associated with significant reductions in expression of osteoblast marker genes (Runx2, alkaline phosphatase, osteocalcin) and the osteoclast marker gene, Trap5. Importantly, in this model, 1D11 did not improve kidney function or reduce serum parathyroid hormone (PTH) levels, indicating that 1D11 effects on bone are independent of changes in renal or parathyroid function. 1D11 also significantly attenuated high-turnover bone disease in the adenine-induced uremic rat model. Antibody administration was associated with a reduction in pSMAD2/SMAD2 in bone but not bone marrow as assessed by quantitative immunoblot analysis. Immunostaining revealed pSMAD staining in osteoblasts and osteocytes but not osteoclasts, suggesting 1D11 effects on osteoclasts may be indirect. Immunoblot and whole genome mRNA expression analysis confirmed our previous observation that repression of Wnt/ß-catenin expression in bone is correlated with increased osteoclast activity in jck mice and bone biopsies from CKD patients. Furthermore, our data suggest that elevated TGF-ß may contribute to the pathogenesis of high-turnover disease partially through inhibition of ß-catenin signaling.

Modeling hepatic osteodystrophy in Abcb4 deficient mice.

Hepatic osteodystrophy (HOD) denotes the alterations in bone morphology and metabolism frequently observed in patients with chronic liver diseases, in particular in case of cholestatic conditions. The molecular mechanisms underlying HOD are only partially understood. In the present study, we characterized the bone phenotypes of the ATP-binding cassette transporter B4 knockout mouse (Abcb4(-/-)), a well-established mouse model of chronic cholestatic liver disease, with the aim of identifying and characterizing a mouse model for HOD. Furthermore, we investigated the influence of vitamin D on bone quality in this model. The bone morphology analyses revealed reduced bone mineral contents as well as changes in trabecular bone architecture and decreased cortical bone densities in Abcb4(-/-) mice with severe liver fibrosis. We observed dysregulation of genes involved in bone remodeling (osteoprotegerin, osteocalcin, osteopontin) and vitamin D metabolism (7-dehydrocholesterol reductase, Gc-globulin, Cyp2r1, Cyp27a1) as well as alterations in calcium and vitamin D homeostasis. In addition, serum RANKL and TGF-ß levels were increased in Abcb4(-/-) mice. Vitamin D dietary intervention did not restore the bone phenotypes of Abcb4(-/-) animals. We conclude that the Abcb4(-/-) mouse provides an experimental framework and a preclinical model to gain further insights into the molecular pathobiology of HOD and to study the systemic effects of therapeutic interventions.